Natriuretic hormone

ABSTRACT

A purified naturally occurring natriuretic compound, identified as Natriuretic Hormone, and a method for isolation of the compound. The natriuretic compound has a molecular weight of about 360, a molecular formula of C 21  H 28  O 5  and with a steroidal nucleus. The compound is useful as a diuretic in the treatment of diseases such as heart disease and hypertension.

This application is a continuation of application Ser. No. 426,497,filed Oct. 23, 1989, now abandoned, which is a continuation ofapplication Ser. No. 217,458, filed Jul. 11, 1988, now abandoned.

FIELD OF THE INVENTION

This invention relates to a compound having a natriuretic effect whichcan be used to increase sodium excretion in man or other mammals.

BACKGROUND OF THE INVENTION

The nature of the system controlling sodium excretion in man and otherterrestrial mammals has been investigated for many years. Until theearly 1960's, sodium excretion in mammals was believed to be controlledby changes in one of two factors: 1) glomerular filtration rate ("GFR");and 2) mineralocorticoid hormone activity. In 1961, however, it wasdemonstrated by DeWardener et al., Clinical Science, Vol. 21, pp.249-258 (1961), that an additional factor, or factors, regulated sodiumexcretion. It was observed that increased sodium excretion (hereinafterreferred to as "natriuresis") occurred in response to extracellularfluid volume expansion in dogs despite constant GFR andmineralocorticoid hormone activity.

Since the observations of DeWardener et al., considerable effort hasbeen employed by researchers in the field to isolate and identify otherfactors involved in the regulation of sodium excretion. In the beliefthat there exists a principal modulator of sodium excretion, a number ofresearchers have pursued a substance referred to as the "natriuretichormone." See, e.g., Bricker, N. S., "The Control of Sodium ExcretionWith Normal and Reduced Nephron Populations: Pre-Eminence of ThirdFactor," Am. J. Med., 43:313 (1967); see also Haber and Haupert,Hypertension, 4:315 (1987).

Prior to the present invention, natriuretic factors acting on theNa/K/ATPase pump had not been isolated in pure form, chemically defined,or synthesized in the laboratory. In fact, there is evidence that morethan one biochemical compound may be responsible for observed effects.The inhibitors of active sodium transport inhibiting fractions studiedmolecular weight of less than 500 Daltons (as indicated by ultrafiltration). Some groups have reported that the compound could be apeptide, but recent studies have not supported the petide nature of thefactor.

Considerable research over the last several decades has focused onadverse effects of a high sodium diet (for example in hypertension) andon the renal retention of sodium in a number of diseases, includingheart disease, liver failure and pre-menstrual syndromes. Diureticagents are widely used today in an effort to prevent or reverse thesepathologic states. However, most potent widely used diuretics not onlyincrease sodium excretion but may also lead to undesirable loss ofpotassium. Unfortunately, the potassium supplements prescribed forreplacement are generally unpalatable, expensive and difficult forpatients to tolerate on a continuing basis.

There thus exists a need for a potent natriuretic compound whichspecifically augments sodium excretion but does not produce the loss ofpotassium. The present invention satisfies this need and providesrelated advantages as well.

SUMMARY OF THE INVENTION

The present invention provides a substantially purified natriureticcompound, termed "Natriuretic Hormone", that increases sodium excretionwithout adversely affecting potassium excretion. The Natriuretic Hormonehas a steroidal nucleus, a molecular weight of 360.4 and a molecularformula of C₂₁ H₂₈ O₅.

In one aspect of the invention, the Natriuretic Hormone is obtained bylyophilizing and reconstituting at a reduced volume with deionized waterthe urine from uremic patients to obtain concentrated samples,separating the concentrated material by gel filtration, subjecting thepost salt peak to reverse-phase high pressure liquid chromatographyusing a pyridinium acetate/methanol buffer. The biologically activematerial activity is eluted with about 45% methanol. The purifiedproduct of the HPLC is then trimethylsilylated and subjected to gaschromatography at elevated temperature. The single product of thischromatography is then hyrolyzed in acid solutions to yield afterevaporation the Natriuretic Hormone.

In another aspect of the invention, the Natriuretic Hormone is used toincrease the sodium excretion in a mammal.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 provides a representation of an ultra violet absorption spectraof a purified active sample as described in Example I.

DETAILED DESCRIPTION OF THE INVENTION

In the course of investigations leading to the present invention, acompound having natriuretic properties was isolated, in substantiallypure form. The compound is termed the Natriuretic Hormone. ThisNatriuretic Hormone can be obtained from a variety of sources, includingthe blood and urine of mammals, preferably humans or dogs andhomogenates of selected tissues such as the hypothalamus. Although thecompound is present in normal human urine, the amount harvested per 24hour urine collection is markedly greater in urine from uremicindividuals.

The Natriuretic Hormone has molecular weight of 360.4, a molecularformula of C₂₁ H₂₈ O₅ and a steroidal nucleus. Additional informationrelating to the physical properties of the Natriuretic Hormone is alsoavailable. It has been isolated as a white powder and exhibits a majorultraviolet absorbance peak at about 220 nm and a broad secondary peakat about 290 nm. The Natriuretic Hormone has been found to be highlyresistant to temperature extremes, maintaining biologic activity asdescribed below after over a year of exposure to minus 80° C. orvigorous boiling. It is water soluble and has also been found to besoluble in certain organic solvents with high dielectric constants,indicating its polar nature. It is eluted from a Sephadex G-25 columnemploying ammonium acetate (NH₄ OAc) buffer, pH 6.8, and appears afterthe salt peak.

The biological activity of the Natriuretic Hormone is markedly decreasedby exposure to 5N HCl. It appears that the reaction taking place is dueto interaction of acid with hydroxyl, carbonyl, or carboxy groups on thesteroidal nucleus, which are known to undergo elimination (e.g.,dehydration), rearrangement (e.g., reverse aldol), and/or hydrolysis(e.g., lactone cleavage) reactions in appropriate circumstances.

Additionally, the Natriuretic Hormone can be further purified using ahigh pressure liquid chromatography (HPLC) column (reverse-phase HPLCusing a C-18 resin column) employing a 0.2m pyridinium acetate buffer(PH 5.5)/methanol gradient, followed by repeated isocratic elution fromthe same column at a methanol concentration of about 40 to 50%,preferably 45%.

As used herein, the term "Natriuretic Hormone" refers to a compoundwhich increases the rate of sodium excretion in at least one mammal uponadministration by inhibiting Na-K-ATPase activity in the nephron. Thenative human Natriuretic Hormone has a molecular weight of 360.4, amolecular formula of C₂₁ H₂₈ O₅ and a steroid nucleus.

The term Natriuretic Hormone refers to both the native hormone and invitro or in vivo modifications which retain natriuretic activity. It isunderstood that limited modifications, substitutions or deletions offunctional groups may be made without destroying the biologicalactivity. Moreover, it will be recognized by those skilled in the artsof steroid chemistry and pharmaceutical preparations that manyderivatives can be made which are biologically and chemically equivalentto, or even more active than, the indicated compound. Examples ofequivalent compounds include esters of acid functions or esters orethers of hydroxylic functions, or common carbonyl derivatives ofcarbonyl functions.

"Substantially pure", when used to describe the state of the NatriureticHormone, denotes the hormone essentially free of proteins, steroids, andother materials normally associated or occurring with NatriureticHormone in its native environment.

As used herein, the term "post salt peak" refers to material eluted froma G-25 Sephadex column which appears immediately after the sodium,potassium, urea and creatinine containing fractions and which hasbaseline conductivity and UV absorbance at 280 nm.

The biological activity of the Natriuretic Hormone can be determined bya number of assay techniques which involve sodium transport in a widevariety of cell types and in a number of animal species. For example,transepithelial sodium transport is inhibited in the isolated urinarybladder of the toad, in the isolated sk the frog and in the isolatedperfused cortical collecting tubule of the rabbit nephron. In all threestructures, inhibition of sodium transport occurs only if theNatriuretic Hormone is added to the blood side of the structure. In theisolated tubule preparation, the addition of surface produces a decreasein sodium efflux (lumen to bath), but there is no effect if it is addedto the lumen surface. There is a simultaneous decrease in thetransepithelial electrical potential difference (P.D.) with the lumenbecoming less positive. In dose response studies, both sodium efflux andP.D. approach zero. As used herein, the term "biologically active"refers to material which results in greater than 20% inhibition of ⁸⁶ Rbuptake as measured by the method of Example II.

In addition, the Natriuretic Hormone produces an increase in sodiumexcretion in the normal Sprague-Dawley rat when fasted but allowed freeaccess to water. It also produces natriuresis in an unanesthetizeduremic rat. When the hormone is infused directly into the renal arteryof the normal rat kidney, natriuresis is moderate. When the equivalentdose is infused into the renal artery of the kidney of a uremic animal,the natriuretic response is markedly increased.

The Natriuretic Hormone inhibits sodium transport by MDBK cells, a cellline originally obtained from bovine renal tubules and maintained usingcell culture techniques. Such a cell line is available from the AmericanType Culture Collection, 12301 Park Lawn Drive, Rockville, Md. 20852,U.S.A., where it is identified by the reference ATCC CCL34 (certifiedcell line). The preferred method of assaying bioactivity according tothe present invention, described in Example II below, employs amicroassay which is based upon the ability of the natriuretic compoundof the present invention to inhibit ⁸⁶ Rb influx by MDBK cells grown inculture. Ouabain (10⁻⁵ M) is used as the reference inhibitor.Natriuretic Hormone also inhibits sodium efflux and influx (inshort-term measurements) as well as ⁸⁶ Rb by the MDBK cell line inculture.

The Natriuretic Hormone of the invention can be used in a number ofclinical and diagnostic contexts. Clinical indications include edematousconditions, such as congestive heart failure, cirrhosis of the liveraccompanied by edema or ascites, nephrotic syndrome, and hypertensiveconditions. Because the compound of the invention is resistant toinactivation under acidic or alkaline conditions, oral ingestion ispossible and preferred. However, administration by other routes, such asintravenous, intramuscular, transdermal and the like, is also effective.

The amount administered at any one time will increase sodium excretionsufficiently to provide a beneficial clinical result. Later doses can beadjusted in accordance with clinical effects of the initial dose. Atypical initial dose would be from about 1-1000 mg in an average human,preferably 10-250 mg, and more preferably 25-100 mg. The total dailydose can consist of a single individual dose or multiple doses given atintervals.

Pharmaceutical preparations can include, in addition to the activecompound, various inert carriers and/or other inactive components suchas moistening agents, flavors, binding agents, and extenders, as well asother compounds having pharmacological activities, such as otherdiuretics which increase the distal delivery of sodium (i.e.,acetazolamide).

The pharmaceutical compositions can take the form of tablets, capsules,injectable solutions and suspensions, oral solutions, and otherformulations intended for pharmaceutical use. For example, a compositionintended for use in a tablet could contain 25 mg active material,calcium stearate, calcium sulfate, microcrystalline cellulose,peppermint oil, polysorbate 80, povidone, and pregelatinized starch.

In addition to use in humans, the natriuretic compound of the inventioncan be used for similar veterinary purposes in domesticated animals,particularly pets with sodium retaining diseases and high bloodpressure.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may be alternativelyemployed.

EXAMPLE I Isolation of Natriuretic Hormone

Twenty four hour urine samples were collected over periods of one to tendays from non-dialyzed patients with serum creatinine concentrations ofover 8mg/dl and/or a serum creatinine clearance of less than 20-25ml/minute. Notation was made of the volume of urine collected each day,and of all medications taken by the patient. Each 24 hour urinecollection was lyophilized to a sludge under reduced pressure andtemperature and reconstituted in 100 ml of deionized water. Thepreparation was then centrifuged at 3000 rpm at 4° C. and filteredthrough fluted filter papers, (Whatmann #1).

In brief, 25 ml aliquots of the concentrated urine samples, equivalentto 6 hour samples of original urine, were applied to individual2.5×95-cm columns packed with Sephadex G-25 (fine grade, Phamacia FineChemicals, Inc., Piscataway, N.J.). Elution was carried out at 4° C. bygravity at a rate of 55 to 65 ml/h with a solution of 10 mM ammoniumacetate at pH 6.8. The effluent solution was collected overnight in18×150-mm glass tubes (12 ml) using an automated fraction collector(model 7000 Ultrorac, LKB Producer AB, Stockholm, Sweden). On the basisof the ultraviolet absorption at 280 nm (LKB Uvicord) and of theelectrical conductivity tracings (model 5300B Conductolyzer with 5312BConductivity cell, LKB Producter AB), the effluent solution and thecontents of the tubes were pooled into several different fractions. Thefractions containing high molecular weight compounds (e.g., proteins),sodium, potassium, urea, and creatinine were discarded. Natriureticactivity was present only in the fractions appearing immediately afterthe salt peak. Only this portion of the effluent solution was thenroutinely prepared by pooling the contents of 10 consecutive tubes (120ml total) starting with the tube in which the specific conductance ofthe eluate had returned to base-line values and lyophilizing thematerial to dryness overnight in a glass container.

The dried eluate was then dissolved in 3.0 ml of distilled water,transferred into a screw cap glass or polyethylene vial, and stored at-80° C. Each milliliter of this solution was equivalent to the volume oforiginal urine excreted in approximately 2 h. Storage of theconcentrated urine samples or of the final fractions for periods of upto several months was found to have no influence on the natriureticactivity of the material.

The standard fractions subjected to assay, whether obtained from uremicpatients or normal subjects, typically contained less than 10 meq/literof sodium and 1 meq/liter of potassium. Differences were not observedbetween fractions from uremic patients and normal subjects for ammonium,urea, and protein concentrations. The mean values were: ammonium, 24.4 ±3.7 meq/liter; urea, 15.8 ± 2.6 mg/100 ml; and protein, 2.0 ± 0.6 mg/ml.

Biologically active fractions, as determined by the method of ExampleII, of post salt peak from the G-25 eluate were applied to a reversephase chromatography column RP-C-18 (Altex Scientific, Berkeley, CA, nowBeckman Instruments, Brea, CA) and eluted with a continuous gradient of0.2M pyridinium acetate (PH 5.5)/methanol (20-60% methanol). Individualtubes were evaporated to dryness employing a "Speed Vacuum" (SavantInstruments, Farmingday, N.Y.), redissolved in deionized water, thentested for biological activity by the method of Example II. Employingthe same column, the combined active fractions were recombined,re-chromatographed two additional times and eluted with 45%methanol/0.2M pyridinium acetate (PH 5.5). After each HPLC run, theresulting active fractions were combined and taken to dryness as above.The fractions were redissolved in deionized water and reassayed forbiological activity.

The ultraviolet absorption spectrum of this material exhibited maxima atapproximately 220 and 290 nm. The material exhibited intrinsicfluorescence in a Beckman Fluorometer, Beckman Instruments, Brea, CA.

The biologically active material was then reacted with a large excess of1:1 pyridine:BSTFA (bistrimethylsilyl trifluoroacetamide) at 50° for 30minutes. The resulting mixture was then applied to a gas chromatograph,using a 15 meter fused silica capillary column (J&W Scientific DB-5-30W)employing a high resolution E-I mass spectrometer as a detector. Theretention time of the single product was 200 to 700 seconds. No otherproducts were detected. The trimethylsilyl groups were hydrolyzed bytreatment with 1N hydrochloric acid for one hour giving, afterlyophilization, a substantially pure white solid, the NatriureticHormone.

EXAMPLE II Microassay For Biological Activity

The natriuretic activity of the Natriuretic Hormone can be ascertainedby its effect on the uptake of ⁸⁶ Rb by MDBK (Malbin Darby Bovine KidneyCell) cultures. Briefly, uptake by the cells of ⁸⁶ Rb (which behavesbiologically as potassium) is inhibited by factors which inhibit theactivity of the enzyme, Na-K-ATPase. Ouabain is a known inhibitor ofthis enzyme and the addition of ouabain to an MDBK cell preparationcontaining ⁸⁶ Rb can result in as much as 90% inhibition of ⁸⁶ Rb intakeby the cells.

Approximately 200,000 MDBK cells were plated in trays containing 12 2mm² wells, and grown for four days at 37° C. in Dulbecco's modifiedEagle media (DMEM) (Gibco #430-2100), to which was added (per liter) 3.7g sodium bicarbonate, 5 mg phenol red, 10% fetal calf serum, 10⁶ unitspenicillin-G sulfate, 0.1g equivalent base streptomycin sulfate, 7.34 mgpolymyxin-B sulfate, and 3.4 mg fungizone and maintained in anatmosphere of 5% CO₂, 95% air. The cells became confluent and present ata concentration of approximately 10⁶ cells per well. To perform theassay, the cells were preincubated for thirty minutes with the testmaterial 50-75 ul in volume which represents at least 2-5 minutes oforiginal urine, in fortified DMEM (500 μl) at 37° C. After thispreincubation, approximately 5×10⁵ cpm ⁸⁶ Rb and an additional 50-75 μlof test material were added to each well and the cells incubated at 37°C. for an additional 15 minutes. The reaction was stopped with 1 ml ofcold PBS and washed twice with 0.5 ml of cold PBS. The washes werediscarded. 0.5 ml of 5% TCA was added to each well and incubated for 10minutes at 37° C., to release radioactivity inside the cells. Thesupernatents from each well were then transferred to a microfuge tube (1ml). A 100 μ1 aliquot (in duplicate) was transferred to scintillationvials containing scintillation fluid (Ready Safe for aqueous samples;Beckman Instruments, Brea, CA.). The ⁸⁶ Rb activity was counted in ascintillation counter (Beckman LS 3-801). The cells were then washedwith PBS and digested overnight at 37° C. in 500 μL 0.1 N NaOH. The nextday, the hydrolyzed cells were analyzed for protein by the microCoomassie method (Chiapelli, F. et al., Analytical Bioch., 94:160 1974),which is incorporated herein by reference. In each set of assays, PBSand 10⁻⁵ M Ouabain were used for negative and positive controls,respectively. Data were recorded as cpm/mg cell protein. Fractions whichinhibited ⁸⁶ Rb uptake by more than 20% were considered active. Mostactive samples inhibited ⁸⁶ Rb uptake by a substantially greaterpercentage. Typical results are shown in Table I.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

                  TABLE I                                                         ______________________________________                                        Each sample run in duplicate and averaged.                                                  cpm/mg                                                                        of protein                                                                            % inhibition                                            ______________________________________                                        Control +PBS    79,692      0%                                                                69,058                                                        Ouabain .sup.10-5                                                                             17,649    77.7%                                                               15,611                                                        G-25 "Post Salt"                                                                              45,592    41.4%                                               Natriuetic      41,596                                                        Activity                                                                      (10 uL)                                                                       (50 uL)         32,336    58.0%                                                               20,803                                                        ______________________________________                                    

We claim:
 1. A method of obtaining substantially purified natriuretichormone comprising a compound characterized by its ability to increasesodium excretion in the urine in mammals without a correspondingincrease in potassium excretion from a mammalian sample containing saidcompound in an aqueous solvent comprising the steps of:(a)pre-concentrating said mammalian sample by removing said solvent fromsaid mammalian sample containing said compound; (b) reconstituting saidpre-concentrated sample to a reduced volume with deionized water; (c)separating a solution of said reconstituted pre-concentrated sample; (d)applying said solution to gel filtration through a Sephadex G-25 columnusing a solution of ammonium acetate as an eluant and retaining onlymaterial appearing immediately after a salt peak to obtain a post-saltpeak material; (e) post-concentrating said post-salt peak material byremoving de-ionized water from said post-salt peak material; (f)reconstituting said post-concentrated post-salt peak material to areduced volume with deionized water; (g) isolating a biologically activefraction from said reconstituted material by repeated reverse-phase highpressure chromatography on a resin column using pyridiniumacetate/methanol as eluant; (h) reacting said isolated biologicallyactive fraction with a trimethylsilylating agent; (i) applying saidtrimethylsilylated biologically active fraction through a gaschromatograph; (j) recovering said gas chromatographed product having aretention time of about 200-700 seconds; (k) subjecting said gaschromatographed product to hydrolysis with acid; and (l) separating saidhydrolyzed product whereby said compound is obtained.
 2. A substantiallypurified natriuretic hormone obtained by said method of claim
 1. 3. Amethod of obtaining substantially purified natriuretic hormone of claim1 wherein further said pre-concentrating step is by lyophilization.
 4. Amethod of obtaining substantially purified natriuretic hormone of claim1 wherein further said post-concentrating step is by lyophilization. 5.A method of obtaining substantially purified natriuretic hormone ofclaim 1 wherein further said mammalian sample is selected from the groupconsisting of blood, urine and tissue.
 6. A method of obtainingsubstantially purified natriuretic hormone of claim 1 wherein furthersaid trimethylsilylating agent is pyridine:bistrimethylsilyltrifluoroacetamide.
 7. A method of obtaining substantially purifiednatriuretic hormone of claim 1 wherein further said hydrolysis with acidis conducted with 1N hydrochloric acid.
 8. A substantially purifiednatriuretic hormone of claim 1 wherein further said trimethylsilylatedgas-chromatographed product has a molecular formula of C₂₁ H₂₅ O₅.3. 9.A substantially purified natriuretic hormone produced by the method ofclaim 1 wherein further said compound(i) has a molecular weight of about360; (ii) has a molecular formula of C₂₁ H₂₈ O₅ ; (iii) has a steroidalnucleus; (iv) is a white powder; (v) exhibits a major ultravioletabsorbance peak at about 220 nm and a broad secondary peak at about 290nm; (vi) is highly resistant to temperature extremes, maintainingbiological activity after over a year of exposure to -80° C. or vigorousboiling; (vii) is water soluble; (viii) is soluble in organic solventswith high dielectric constants; (ix) exhibits a markedly lowerbiological activity when exposed to 5N HCl; (x) inhibits transepithelialsodium transport(1) in the isolated urinary bladder of the toad; (2) inthe isolated skin of the frog; (3) in the isolated perfused corticalcollecting tubule of the rabbit nephron; (xi) produces an increase insodium excretion in the normal Sprague-Dawley rat when fasted butallowed free access to water; (Xii) produces natriuresis in anunanesthetized uremic rat; and (xiii) inhibits sodium transport by MDBKcells.
 10. A pharmaceutical composition useful for treating mammals withedematous conditions or hypertension comprising an amount of thecompound of claim 9 effective to treat said edematous conditions orhypertension and a pharmaceutical acceptable carrier.